Sleeping with the Cavefishes

As a graduate student, I would give my right arm to be a fully functioning human being with little to no sleep. Alas, even Aristotle in 350 BCE observed a seemingly simple truth -- all animals sleep. Much to the frustration of sleep scientists, we still do not fully understand why we need sleep or why there is so much variation between species in sleep behavior. We are, however, beginning to gain an understanding of what may be regulating sleep and how it may have evolved over time, in some cases even making use of unusual model organisms such as Astyanax mexicanus, or the Mexican cavefish.

The Mexican cavefish is a unique model because it exists in two varieties: an eyeless, cave-dwelling fish that has developed characteristics that allow it to survive in its distinctive environment, and a still existing ancestral surface-dwelling fish that lives in superficial rivers. The cavefish dwells in multiple caves found in the Northern region of Mexico and many of these geographically isolated cavefish populations have independently evolved a unique and enviable trait: they require very little sleep. Recently, researchers from Florida Atlantic University sought to investigate the underlying neural mechanisms responsible for the development of this sleep loss behavior, focusing on the role of Hypocretin/Orexin (HCRT), a neuropeptide expressed in the hypothalamus that regulates wakefulness.

(A) Representative images of surface fish and Pachón cavefish (B) Sleep duration is significantly reduced in Pachón cavefish compared to surface fish (Unpaired t-test, t = 5.56, n = 26, p<0.0001). (C) Expression of hcrt normalized by GAPDH in adult whole-brain extracts is significantly enhanced in Pachón cavefish compared to surface fish. Figure taken from Jaggard et al., 2018. 

The researchers combined behavioral and molecular techniques to determine if HCRT activity is altered in cavefish. They tracked the sleep of the surface- and cave-dwelling fish over a period of 24 hours, and confirmed earlier findings that surface fish sleep significantly more (approximately 6 hours/day) than cavefish (between 1-2 hours/day). The following day, they measured the amount of HCRT gene expression in the brains of the surface and cavefish that had been sleep recorded. They found that the cavefish had more than 3 times the amount of HCRT compared to the surface fish. This gave evidence that cavefish have elevated expression of HCRT and a decrease in sleep but did not give conclusive evidence that HCRT was driving the sleep loss behavior.

To determine whether HCRT was necessary for the sleep loss behavior, the investigators inhibited HCRT function by blocking the HCRT receptor with a drug called TCSOX229. When HCRT function was inhibited, cavefish sleep significantly increased, however not to the same amount of sleep as the surface fish. To further validate these findings as well as to determine the developmental component to sleep regulation, the researchers also conducted this experiment using larval aged fish and found that TCSSOX229 and other HCRT inhibitors also promoted increased sleep behavior. Sleep in the surface fish remained unaltered when given these drugs suggesting that cavefish have a heightened sensitivity to changes in HCRT activity.

Finally, the researchers looked at external factors that could be impacting this sleep-loss behavior. Because of the isolated cave environment, cavefish have developed an increased number of mechanoreceptors, which are specialized cells that respond to pressure, vibration, and other tactile information. This increase in mechanoreceptors, which form a lateral line down a fish’s body, makes the cavefish more sensitive to movements in the water. It is possible to kill these cells using a drug called gentamicin, and it has been previously shown that exposure to gentamicin increases sleep in the cavefish. When the researchers exposed both groups of fish to gentamicin, they found that HCRT expression significantly decreased in cavefish but remained unchanged in surface fish. This provides evidence that suggests that their increased wakefulness is promoted by the heightened level of stimulation and that sensory gating plays a strong role in sleep regulation.

The heightened sensitivity to their surroundings is likely an adaption to aid in food foraging, and so another factor they chose to investigate was starvation. Most of the year, the cavefish live in a food-sparse environment with the exception of a brief “rainy season” that can flood outside nutrients into the cave. A persisting theory in the field is that when food is limited, sleep will decrease so that the animal can spend more time foraging. When the surface and cavefish were starved for several days, they found that the cavefish not only change their sleep behavior but also increased their HCRT expression. The surface fish however, showed the opposite effect, perhaps due to the fact that food is always readily available in their environment. This suggests that the elevated HCRT may also be closely linked to adaptive foraging strategies developed by the cavefish.

This study nicely uncovers the role that hypocretin plays in regulating sleep behavior. Furthermore, the researchers draw attention to the complexity of sleep behavior and its entanglement in many other physiological behaviors such as sensory integration and foraging. Their findings provide strong evidence that this remarkable sleep-loss behavior is driven by a heightened production of this wake-promoting neuropeptide and give great insight into the types of factors that can impact the regulation of HCRT. 

Reference:

James B Jaggard, Bethany A Stahl, Evan Lloyd, David A Prober, Erik R Duboue, and Alex C Keene (2018) Hypocretin underlies the evolution of sleep loss in the Mexican cavefish. eLife 2018;7:e32637. PMCID: PMC5800846